Project Summary Viruses have a limited genome and, therefore, must commandeer host cell processes in order to mount a productive infection. One way viruses manipulate host processes is by hijacking the cellular ubiquitin system in order to redirect ubiquitination of host proteins. Ubiquitination is a protein post-translational modification in which an E3 ubiquitin ligase enzyme catalyzes the attachment of a ubiquitin molecule to a protein substrate. The most well understood outcome of ubiquitination is targeting the substrate for proteasomal degradation. However, it has become increasingly appreciated that ubiquitination can also direct functional outcomes other than degradation, such as altering protein function, location or interactions. It is well known that viruses redirect cellular ubiquitination in order to degrade host anti-viral factors. However, there is a gap in knowledge as to whether and how viruses use alternative functions of ubiquitin to control cellular processes during infection. Human Adenovirus (AdV) offers a compelling system in which to study viral-mediated ubiquitination because it is known to redirect ubiquitination of host proteins to support infection but the range of targeted substrates is unknown. AdV encodes two viral proteins, E1B55K and E4orf6, that complex with host proteins to form a viral E3 ubiquitin ligase. AdV lacking a functional E1B55K/E4orf6 ligase exhibits RNA processing deficiencies. However, the known substrates of the viral ubiquitin ligase can not explain the mechanistic link between the ligase and viral RNA processing. I propose that there are unknown substrates of the E1B55K/E4orf6 ubiquitin ligase that impact viral RNA processing. I analyzed ubiquitin-proteomics datasets to identify unknown E1B55K/E4orf6 substrates. The predicted substrates are enriched for RNA-binding proteins, which are potentially important to describe the ligase function in viral RNA processing. Furthermore, the RNA-binding substrates are ubiquitinated but not decreased in abundance, suggesting that this analysis identifies the first known examples of AdV non-degradative ubiquitination. Additional preliminary data suggest that deletion of the E1B55K/E4orf6 ligase decreases viral RNA splicing efficiency. Therefore, I propose to define the role of AdV- mediated ubiquitination in viral RNA processing through two specific aims that combine experimental and bioinformatics approaches. I will 1) examine the protein-protein and protein-RNA interactions of the RNA- binding substrates of E1B55K/E4orf6 and 2) examine the impact of E1B55K/E4orf6 on viral RNA splicing. This study will identify novel E1B55K/E4orf6 substrates that can link the function of the viral ligase to viral RNA processing. Further, novel mechanisms by which viruses use ubiquitination to manipulate cellular processes will be revealed. Accomplishing the proposed aims will provide me training in experimental methods to study host-pathogen interactions and strengthen my background in bioinformatics. Ultimately, this fellowship will provide the foundation for my future as a principal investigator leading a lab that combines bioinformatics and experimental approaches to address important questions in host-pathogen interactions.